Osseointegration in additive-manufactured titanium implants: A systematic review of animal studies on the need for surface treatment

The objective of the systematic review is to find an answer to a question: “Do surface treatments on titanium implants produced by additive manufacturing improve osseointegration, compared to untreated surfaces?“. This review followed the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA 2020) and was registered in the International Prospective Register of Systematic Reviews (PROSPERO) (CRD42022321351). Searches were performed in PubMed, Scopus, Science Direct, Embase, and Google Scholar databases on March 22nd, 2022. Articles were chosen in 2 steps by 2 blinded reviewers based on previously selected inclusion criteria: articles in animals that addressed the influence of surface treatments on osseointegration in implants produced by additive manufacturing. Articles were excluded that (1) did not use titanium surface, 2) that did not evaluate surface treatments, 3) that did not described osseointegration, 4) Studies with only in vitro analyses, clinical studies, systematic reviews, book chapters, short communications, conference abstracts, case reports and personal opinions.). 1003 articles were found and, after applying the eligibility criteria, 17 were used for the construction of this review. All included studies found positive osseointegration results from performing surface treatments on titanium. The risk of bias was analyzed using the SYRCLE assessment tool. Surface treatments are proposed to promote changes in the microstructure and composition of the implant surface to favor the adhesion of bone cells responsible for osseointegration. It is observed that despite the benefits generated by the additive manufacturing process in the microstructure of the implant surface, surface treatments are still indispensable, as they can promote more suitable characteristics for bone-implant integration. It can be concluded that the surface treatments evaluated in this systematic review, performed on implants produced by additive manufacturing, optimize osseointegration, as it allows the creation of a micro-nano-textured structure that makes the surface more hydrophilic and allows better contact bone-implant.


Introduction
The additive manufacturing (AM), like software aid (CAD), technique in the manufacture of implants can allow the manufacture of customized three-dimensional (3D) metallic devices, with controlled surface characteristics, custom geometries and different levels of porosity [1][2][3][4]. This justifies the investment to increase the use of this technique in the manufacture of implant parts that require more complex structures, as rougher surfaces, adequate rigidity and good stress transfer [5].
Additive manufacturing in metallic implants is performed by selective laser fusion (SLM) and electron beam fusion (EBM) processors [1,[6][7][8]. Both are made using rapid prototyping technology, where the metal alloy powder is melted by exposing the SLM or EBM process to reproduce, the final model of the implant [1,[9][10][11]. The SLM uses a high power laser beam, for the melting of the metal alloy powder, simultaneously with this melting process, it is released an inert gas in an attempt to protect the particles from overheating [1,5,7].The EBM performs the melting by an electron beam that generates a lower temperature than the SLM, in addition, this process is carried out in a closed chamber, with high vacuum levels, protecting materials from oxidation [1,7].
These methods can reduce the waste of material, time, and money compared to the conventional machining technique, in addition, they aim to create varied structures, with uniform density distribution and control of the quality and quantity of pores on the surface [4,9,10]. This last advantage in AM, since these pores are determinant for the mechanical properties and biological that precede the osseointegration [9,12,13].
It is known, however, that surface treatment techniques, such as TiO2 nanotubes, hydroxyapatite, collagen type I, calcium phosphate, tantalum, polydopamine, acid attack and anodic oxidation, Hydrofluoric/nitric acid, inorganic chemical oxidation, sodium hydroxide, chitosan and UV photofunctionalization, despite their advantages, deserve care, mainly due to the high degree of contamination and toxic elements that can be introduced into titanium, which goes against the principle that the ideal surface for better mechanical stability and osseointegration capacity is the one with the highest purity of titanium oxide and adequate roughness, which thus favor the activities of osteogenic cells and absorption of plasma proteins that regulate osteoblastic adhesion in the biomaterial and thus configure themselves as fundamental characteristics for the osseointegrative process [10,14,15].
Although studies suggest better biological in dental implants obtained by AM [1,3,6,8,16,17]. While articles in the literature evaluate surface properties related to the application of the coating, the originality and relevance of the present systematic review is to evaluate how this surface treatment, whether chemical, electrochemical, or mechanical, influences osseointegration and to understand how this reflects in the best possible way. Bone-implant contact and the absorption of cells that are optimizing this process. Thus, the objective of this systematic review was to present the current state of the art on the need to carry out surface treatments in titanium dental implants produced by additive manufacturing technique. For this, this review was built from the null hypothesis that the performance of surface treatment on titanium dental implants does not influence osseointegration.

Protocol
The present systematic review sought to answer the question: "Do surface treatments on titanium implants produced by additive manufacturing improve osseointegration, compared to untreated surfaces?" This review was constructed following the preferred reporting items for the systematic review and meta-analysis (PRISMA 2020) [18]. In addition, the protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) (CRD42022321351). The search strategy used for a population, intervention, comparison, outcome, and study design (PICOS) is described in Table 1.

Eligibility criteria
This review included articles that addressed the influence of surface treatments on osseointegration in manufactured implants. For that, articles with in vivo analyses in animals were used. After defining the exclusion criteria, articles that did not use titanium surfaces or that used titanium surfaces that were not produced by additive manufacturing, that did not evaluate surface treatments and that did not describe osseointegration were removed. Studies with only in vitro analyses, clinical studies, systematic reviews, book chapters, short communications, conference abstracts, case reports, and personal opinions were also excluded.

Search strategy
The search strategy was applied to the following electronic databases: PubMed, Scopus, Science Direct, Embase, and Google Scholar (Supplementary Table 1) in March 22nd, 2022. This search was performed and the selected studies, based on the eligibility criteria, are published between the years 2015-2022. In addition, an additional search was performed in the reference and citation lists of the included articles to find new possible inclusions.
The first reading of the articles was performed by 1 author (J.V⋅C⋅N.). The findings were attached to the Rayyan digital platform and then evaluated by 2 independent authors (J.V⋅C⋅N., M.L.C⋅V.) who were responsible for analyzing the articles according to the preestablished inclusion and exclusion criteria. The other studies were read in full. Conflicting results were resolved by the third author (A.C.R.). Data extraction from the article was done through a table with the following topics: Author/year; Goal; Evaluated Animals;

Risk of bias assessment
For this evaluation, the tool for animal studies called SYRCLE [19] was used (Supplementary Table 2). Based on the analysis of these criteria, the article is classified as having a low, high, or uncertain risk of bias. When using this tool, it is not recommended to perform calculations and score charts per study.
From the application of the SYRCLE tool, it was observed that in all articles included there were: similar basic characteristics between the groups; concealed allocation to different groups during the study; the case and control groups were randomly distributed among the housing being exposed to the same conditions; random assessment of the outcome of the case and control groups; all results were treated completely; protocol availability and reporting of all results; no other sources of bias were presented.

Certainty of the evidence
The certainty of the evidence for each outcome was calculated by the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. In this assessment, two independent authors (J.V⋅C.N. and M.L.C⋅V.) analyzed the certainty of evidence. Initially, the results are considered with high-quality evidence and the downgrade of the evidence is given by limitations, inconsistencies, indirectness, imprecision, and publication bias criteria. Disagreements were resolved by consensus. Based on the mentioned criteria, the certainty of each piece of evidence was classified as high, moderate, low, and very low [20]. Fig. 1 addresses the strategy used to select the studies. In the first search, 1003 articles were found, of which 91 were excluded due to duplication. After reading the title and abstract with the application of inclusion and exclusion criteria, 17 studies were selected for a full reading. After reading in full, the 17 articles were included for analysis and discussion in this review.

Search results
Performing a meta-analysis was not possible due to the lack of homogeneity of results found in the included articles. Thus, the results were based on a descriptive analysis of the data.

Grading the evidence
For the certainty of the evidence of the selected studies, the GRADE tool was applied. The studies included in this review are randomized pre-clinical studies, the evaluation of which is initiated at a high level of evidence, and as the gradation of quality is performed, this level can be lowered. However, the factors involved in the quality of evidence, such as the risk of bias, inconsistencies, indirect evidence, and imprecision in the results, maintained the certainty of the evidence of the results found at a high level. (Supplementary Table 3). Table 2 provides information regarding the included studies, based on the eligibility criteria, which are published in the years 2015-2022.
Shu et al. [29] noticed a superior osseointegration and gene expression in these coated manufactured implants [29]. Xiu et al. [32] noticed early osteogenesis osseointegration, due to improvements in cellular functions and the higher protein content found [32]. Finally, Yin et al. [34] noticed resulted in greater uptake of plasma proteins, differentiation and adhesion of osteogenic cells that accelerated the osseointegration process [34].

Discussion
This systematic review addressed studies that evaluated the influence of surface treatments on titanium dental implants produced by AM on osseointegration, in the search to elucidate whether these treatments can maximize bone-implant contact and stimulate adhesion and absorption of related proteins. To this phenomenon. The research included contradicted the null hypothesis and indicated that, even in implants produced by additive manufacturing, surface treatments remain essential for the osseointegration process [4,5,9,11,12,[21][22][23][24][26][27][28][29][30][32][33][34].
In the selected articles, there was a great variety in the methodologies of surface treatments addressed that confuse the scientific and clinical community about the real need for these in titanium implants produced by AM since for each type of treatment, different topographic micro characteristics can be achieved and thus different results to osseointegration can be achieved [4,5,9,11,12,21-24, 26-30,32-34].
Several substances and techniques for performing surface treatments are found in the literature, being described as mechanical, such as machining and abrasive blasting, chemical, such as oxidation and acid conditioning, and physical, such as plasma spray [35]. Despite being different techniques, they all revolve around a central objective: to change the microtopographic properties of the surface and favor the biological phenomena that result in osseointegration.
The studies addressed in this review used different surface treatment techniques on implants produced by additive manufacturing to evaluate osseointegration [4,5,9,11,12,[21][22][23][24][26][27][28][29][30][32][33][34]. Regardless of the technique used, it was possible to infer that the surface treatment is of fundamental importance in the osseointegration process since it allows the creation of a micro-nano-textured structure that makes the surface more hydrophilic and allows better bone-implant contact [34] and that drives an improvement in the osteoconductive properties of the surface that results in the acceleration of adhesion and absorption of bone cells and proteins involved in the osseointegration process and bone neoformation. In addition, it is noted that the application of surface treatment improves the amount of residual particles on the surface, hinders adhesion and biofilm formation, in addition to boosting mechanical properties [9,11,27]. Performing surface treatments represent an effective strategy to reduce the risk of post-surgical inflammatory reactions [27] and thus results in a faster and more uniform bone growth pattern around the entire implant, Xiu et al. [32] since the surface finish allows a superior gene expression when compared to implants without treatment [32].
From these findings observed in the articles included and mentioned above, it is possible to perceive that the surface treatment, regardless of the technique or substance used, produces a more favorable surface for osseointegration, making it necessary even in implants produced by additive manufacturing. However, for this osseointegration phenomenon to occur, it is necessary to understand that it does not depend only on the natural biological behavior of the host, but also on the microstructure and chemical composition of the implant surface [32,36].
When considering the current state of the art regarding the additive manufacturing technique for the manufacture of titanium implants, it is noted the promotion of good macrostructural regulation, such as the control of porosity on the surface, which allows for better vascularization, formation of anchorage to bone tissue, differentiation and growth of osteoblastic cells that favor bone fixation To investigate osseointegration in implants manufactured from Ti-6Al-4V, with or without post-processing of HA and collagen type I in an in vivo model.
Ti-6Al-4V interconnected porous structure, with a solid central core and a porous outer layer.  To assess whether nanotopographic modifications in manufactured implants improve osteogenic differentiation and osseointegration.
Ten female SD Rats weighing around 280 g.
Scanning electron microscope; X-ray diffractometer; Wetability; X-rays with energy dispersion spectrometry; Alkaline Phosphate Activity Assay; qRT-PCR; Alizarin Red A Staining Assay; Surface modification in manufactured implants favors osseointegration in the early stages. It is noticed that these implants can replace conventional implants.
(continued on next page) J.V. Calazans Neto et al.  [9,10,[26][27][28], however, the implants produced by this technique still seems to need greater control of the microstructural characteristics responsible for the biological response and osseointegration. Failure to carry out the surface treatment, for example, can facilitate the release of dust particles that are not fused or poorly adhered to the implant surface at the implantation sites and increase the risk of local inflammatory processes or even induce failure points in the implant [1,23,32]. Therefore, several surface modifications are being studied in titanium dental implants produced by additive manufacturing as evaluated in the included articles, to improve their performance and osseointegration capacity [32]. The articles included do not present financial interests or personal relationships that constitute a conflict of interest and may have influenced the result of the work. In the construction of this manuscript, some limitations that made the review process difficult were found, the main one referring to the literary gap on the surface characteristics of titanium implants produced by additive manufacturing. In addition, a limitation in the evidence of the evaluated experimental studies was noted. However, it is possible to infer that the performance of surface treatments on these pieces are of fundamental importance to guarantee biological and microbiological responses that favor the osseointegration of these implants, thus better clinical responses will be obtained, which result in an acceleration of treatment completion and rehabilitative success, with greater patient satisfaction. Finally, the importance of more clinical and experimental studies on the subject is highlighted, with better standardization of methodology, reproducibility, and greater control for greater homogeneity in the results found.

Conclusion
Based on the results of this systematic review, it was possible to reach the following conclusions.
1. Although the additive manufacturing technique aims to produce surfaces with important characteristics and favorable to osseointegration, all included studies determined that the performance of surface treatment, regardless of the chosen technique, is necessary to bring about significant improvements in osseointegration; 2. The methodological heterogeneity of the surface treatments found in the included studies, represents the need to carry out more indepth studies to determine which surface treatment, associated with additive manufacturing, can offer better results to osseointegration.

Author contribution statement
João Vicente Calazans Neto: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Andréa Cândido dos Reis and Mariana Lima da Costa Valente Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Data availability statement
Data included in article/supplementary material/referenced in article.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2023.e17105.   High certainty: we are very confident that the true effect lies close to that of the estimate of the effect. Moderate certainty: we are moderately confident in the effect estimate: the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different. Low certainty: our confidence in the effect estimate is limited: the true effect may be substantially different from the estimate of the effect. Very low certainty: we have very little confidence in the effect estimate: the true effect is likely to be substantially different from the estimate of effect.